Nanopore functionality control

ABSTRACT

A method is provided of controlling the functionality of a substrate containing at least one nanopore. The method includes the steps of: introducing to the substrate a solution containing a molecular construct having a body formation which defines an aperture and a tail formation extending from the body formation; applying a potential difference across the substrate to thread the tail formation through the nanopore thereby docking the molecular construct to the substrate with the aperture aligned with the nanopore such that the sleeve formation lines the nanopore; and expelling the molecular construct from the substrate by varying the potential difference. A DNA construct for docking to a substrate having a nanopore is also provided, the construct having a body formation which defines an aperture, and a tail formation extending from the body formation for threading through the nanopore to dock the construct to the substrate with the aperture and nanopore in alignment.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/361,381, filed May 29, 2014, which is a nationalstage application under 35 U.S.C. 371 and claims the benefit of PCTApplication No. PCT/GB2012/053033 having an international filing date ofDec. 6, 2012, which designated the United States, which PCT applicationclaimed the benefit of Great Britain Application No. 1120910.3 filedDec. 6, 2011, the disclosure of both the above-identified applicationsare incorporated herein by reference.

The present invention relates to a method of controlling thefunctionality of a substrate containing at least one nanopore and a DNAconstruct for insertion into a nanopore.

Single nanopores in insulating barriers are under investigation assensors for single molecules in solution¹. Since the first experimentson DNA and RNA translocations by Kasianowicz et al.² research activitiesin nanopore sensing have grown rapidly. Single solid-state nanoporesproduced by silicon nanotechnology and ion beam milling were firstemployed for ionic current-based detection of DNA by Li et al.³. DNAfolding detection with nanocapillaries⁴, the development of alternativefabrication protocols like transmission-electron drilling of solid-statenanopores⁵, or the combination of nanopores with other single-moleculetechniques like optical tweezers⁶, magnetic tweezers⁷ or single moleculefluorescence⁸ has given the field new impetus.

In a typical application, the nanopore separates two chambers containingan aqueous solution of positive and negative salt ions. Additionally,one of the reservoirs contains a molecule of interest. When a potentialdifference is applied across the membrane an ionic current can bemeasured as ions pass through the nanopore. Molecules translocatingthrough the nanopore transiently block the current. The change incurrent and dwell time as the molecule passes through the nanopore canbe measured with a sensitive current detector. The current tracesrecorded can then be analysed to infer molecular properties.

Two remaining challenges are the control of the exact shape of nanoporesand the analyte-specific modification of their cavities. Coating withlipid bilayers⁹ or polymers¹⁰ provides means of modifying the surfaceproperties of solid-state nanopores, but the creation of fixedstructures with nanometer scale control of geometry and positioning offunctional chemical motifs has yet to be demonstrated. The recentcombination of hybrid biological and solid-state nanopores¹¹ crossed theboundary between nanopores extracted from living organisms and thosederived from silicon nanotechnology. While this achievement allows forthe design of nanopores adapted for molecular sensing¹² andsequencing¹³, protein nanopores have fixed diameters which are often inthe single nanometer range. This currently limits the range of analytesto unfolded proteins or single-stranded DNA chains.

It would be desirable to have a method for forming easily tunablenanopores with diameters from 1 nm to 100 nm that could therefore beapplied to a much wider range of analytes. Further, as solid-statenanopores can be high cost items which may be problematic to manufacturereproducibly (particularly when the nanopores have diameters of 20 nm orless), it would be desirable to improve the usable life and/orreusability of such nanopores. The present invention is at least partlybased on a realisation that control of nanopore size and functionalityin a hybrid nanopore can be achieved by the application of a potentialdifference.

Accordingly, in a first aspect, the present invention provides a methodof controlling the functionality of a substrate containing at least onenanopore, the method including the steps of:

-   -   introducing to the substrate a solution containing a molecular        construct having a body formation which defines an aperture and        a tail formation extending from the body formation;    -   applying a potential difference across the substrate to thread        the tail formation through the nanopore thereby docking the        molecular construct to the substrate with the aperture aligned        with the nanopore; and    -   expelling the molecular construct from the substrate by varying        the potential difference.

Thus, advantageously, the molecular construct can be used to control thefunctionality of the nanopore (e.g. by controlling the construct's size,geometry, chemical functionality, etc. particularly at the aperture),but the construct can be removed, allowing the substrate e.g. to bere-used for a different analyte.

The method of the first aspect may have any one or, to the extent thatthey are compatible, any combination of the following optional features.

The variation of the potential difference may typically include areversal of the potential across the substrate.

The method may include a further step of flushing the expelled molecularconstruct away from the substrate after its expulsion, for example bythe use of an appropriate micro-fluidics arrangement. Indeed, such anarrangement can also be used to introduce the solution containing themolecular construct to the substrate in the first place.

The method may include further steps after the expelling step of:introducing to the substrate a solution containing a different molecularconstruct having a body formation which defines an aperture and a tailformation extending from the body formation; and re-applying a potentialdifference across the substrate to thread the tail formation through thenanopore thereby docking the different molecular construct to thesubstrate with the aperture aligned with the nanopore. In this way, thefunctionality of the nanopore can be controllably and repeatablymodified. For example, a range of different molecular constructs can besuccessively docked to the substrate to make the nanopore functionallysensitive to a corresponding range of different analytes.

In a second aspect, the present invention provides a method of detectingthe presence of an analyte in a solution, the method including the stepsof:

-   -   (a) providing a substrate containing at least one nanopore;    -   (b) introducing to the substrate a solution which may contain        the analyte, and a molecular construct having a body formation        which defines an aperture, and which has a one or more binding        sites for the analyte at the aperture;    -   (c) applying a potential difference across the substrate to dock        the molecular construct to the substrate with the aperture        aligned with the nanopore, and measuring the corresponding        change in ionic current through the nanopore;    -   (d) comparing the measured change in ionic current to a        reference change in ionic current; and    -   (e) determining that the analyte is present in the solution when        the measured change in ionic current differs from the reference        change in ionic current by more than a predetermined amount.

Thus the analyte, when present, binds to the molecular construct in thesolution and, because the one or more binding sites are at the aperture,can thereby fully or partially block the aperture. When the molecularconstruct docks to the substrate, the blockage affects the ability ofions to pass through the nanopore and hence affects the measured changein ionic current.

Advantageously, analyte specificity can thereby be provided by themolecular construct. Thus if a further solution contains a moleculewhich is of similar size to the analyte but which does not bind to themolecular construct, applying steps (b) to (e) to both solutions shouldallow a user to distinguish between the solution containing the analyteand the further solution.

The method of the second aspect may have any one or, to the extent thatthey are compatible, any combination of the following optional features.

The applying step (c) may include repeatedly performing the sub-stepsof:

-   -   (i) applying the potential difference across the substrate to        dock the molecular construct to the substrate with the aperture        aligned with the nanopore, and measuring the corresponding        change in ionic current through the nanopore; and    -   (ii) expelling the molecular construct from the substrate by        varying (e.g. reversing) the potential difference; and    -   wherein, in the comparing step (d), the measured change in ionic        current is an average measured change derived from the repeated        measurements of sub-step (c-i).

Repeating the measurements can help to improve detection accuracy.

Typically, the measured change and the reference change are decreases inionic current.

The reference change can conveniently be the change in ionic currentthat would have occurred if there had been no analyte in the solution.Indeed, the method may further include the steps of:

-   -   (A) providing a substrate containing at least one nanopore;    -   (B) introducing to the substrate a solution containing the        molecular construct in the absence of the analyte; and    -   (C) applying a potential difference across the substrate to dock        the molecular construct, in the absence of the analyte, to the        substrate with the aperture aligned with the nanopore, and        measuring the corresponding change in ionic current through the        nanopore; and    -   wherein, in the comparing step (d), the reference change in        ionic current is the change in current measured at step (C). The        solution of step (B) preferably has the same ionic strength and        pH as the solution of step (b). For example, it may contain an        identical concentration of the same dissolved salt and be        identically buffered.

The applying step (C) may include repeatedly performing the sub-stepsof:

-   -   (i) applying the potential difference across the substrate to        dock the molecular construct, in the absence of the analyte, to        the substrate with the aperture aligned with the nanopore, and        measuring the corresponding change in ionic current through the        nanopore; and    -   (ii) expelling the molecular construct from the substrate by        varying (e.g. reversing) the potential difference; and    -   wherein, in the comparing step (d), the reference change in        ionic current is an average measured change derived from the        repeated measurements of sub-step (C-i).

The substrate of step (A) can be a different but equivalent substrate tothe substrate of step (a). Steps (A) to (C) can then be performedbefore, during or after the performance of steps (a) to (c).

Another option, however, is for the substrate of step (A) to be the samesubstrate as the substrate of step (a). Steps (B) to (C) can then beperformed before or after the performance of steps (b) to (c). However,whichever is performed first, the method may include a further step offlushing the first solution away from the substrate after expulsion ofthat solution's molecular construct, and before the introduction of thesecond solution. Again, the flushing can be performed by the use of anappropriate micro-fluidics arrangement. The arrangement can also be usedto introduce the first and second solutions to the substrate.

The analyte may be an antibody.

The molecular construct may further have a tail formation extending fromthe body formation, the tail formation being threaded through thenanopore to dock the molecular construct to the substrate when thepotential difference is applied across the substrate.

The method of the first or second aspect may have any one or, to theextent that they are compatible, any combination of the followingoptional features.

The substrate may have plural nanopores, and the apertures of respectivemolecular constructs may be aligned with the nanopores by theapplication of the potential difference across the substrate. Likewise,the plural molecular constructs may be expelled from the substrate byvarying the potential difference.

The substrate may be a silicon-nitride or silicon-oxide substrate, e.g.with one or more electron or ion beam drilled nanopores.

Preferably, the body formation has a docking surface which contacts thesubstrate when the construct is docked thereto, at least the dockingsurface being hydrophilic. In a typically aqueous solution, this canhelp to prevent the body formation from binding too tightly to a matingsurface of the substrate, whereby variation of the potential differencecan more easily expel the molecular construct from the substrate. Incontrast, the α-hemolysin protein pore disclosed by Hall et al.¹¹ ishydrophobic.

Preferably, the body formation can include or be a sleeve formationwhich defines the aperture, the application of the potential differencecausing the molecular construct to dock to the substrate such that thesleeve formation lines the nanopore. The above-mentioned docking surfacecan include or be the external surface of the sleeve formation.

Preferably, the molecular construct is a DNA construct. Advantageously,DNA is hydrophilic, which, as discussed above, promotes theexchangeability of the construct. Further, the DNA construct canconveniently be made by DNA-based self-assembly or “origami” techniques.DNA-based self-assembly¹⁴ employs the programmability of DNA sequencesto build rationally designed objects of ever increasing complexity.Owing to the development of DNA origami¹⁵ it is now possible to designand fabricate almost arbitrary nanosized shapes¹⁶. The origami methodtypically uses a 7-8 kb long m13mp18-based single-strand as a scaffoldfor the assembly of hundreds of distinct synthetic staple strands. Eachof these staples can potentially be extended with a nucleotide (nt)sequence of interest or a wide range of chemical modifications¹⁷. Inaddition, the use of DNA origami allows for the addition of functionalchemical groups, fluorophores, gold nanoparticles etc. at sub-nanometreposition accuracy by employing modifications to the oligonucleotidestaple strands used to fold the long DNA single strand²⁵. Thesemodifications have the potential for the integration of DNA origami andnanopores with techniques such as fluorescence detection and Ramanspectroscopy. Thus DNA is well suited for the synthesis of programmableconstructs with chemically adjustable cavities and custom tailoredgeometries.

Indeed, in a third aspect, the present invention provides a DNAconstruct (e.g. a DNA origami construct) for docking to a substratehaving a nanopore, the construct having a body formation which definesan aperture, and a tail formation extending from the body formation forthreading through the nanopore to dock the construct to the substratewith the aperture and nanopore in alignment. The body formation caninclude or be a sleeve formation which defines the aperture and whichlines the nanopore when the construct is docked to the substrate. Theconstruct can include functional chemical groups, fluorophores, and/orgold nanoparticles.

Further, in a fourth aspect, the present invention provides the use ofDNA origami to form the DNA construct of the second aspect.

Further optional features of the invention will now be set out. Theseare applicable singly or in any combination with any of the aboveaspects of the invention.

The nanopore preferably has a diameter of 100 nm or less, and morepreferably of 50 or 20 nm or less. Nanopores of 20 nm diameter or lessare generally particularly difficult to produce reproducibly, or indeedat all. Thus the use of the molecular construct in relation to thenanopores of this size can be particularly beneficial, allowing the samenanopore to be used for repeatedly, e.g. for different analytes. Thisnot only helps to relieve the burden of producing numbers of nanoporesin the first place, but allows the same nanopore to be used forcomparative testing.

The nanopore preferably has a diameter of 1 nm or more, and morepreferably of 2 or 5 nm or more.

Further optional features of the invention are set out below.

Embodiments of the invention will now be described by way of examplewith reference to the accompanying drawings in which:

FIG. 1: DNA origami design. (a) Schematic representation of the DNAorigami structure with the double stranded overhang. Double helices arepresented as rods. (b) Top: Side view of the nanopore section of the DNAorigami structure with helices shown as cylinders. The overall length ofthe nanopore is 150 bp (51.0 nm) with the smallest opening of 7.5×7.5nm². Length A=40 bp (13.6 nm), length B=48 bp (16.3 nm). Bottom: Topview of the structure, indicating the square shape with 11 by 11 heliceswith a total area of 27.5×27.5 nm². (c) Transmission electron microscope(TEM) images of assembled DNA origami constructs negatively stained withuranyl acetate. (d) TEM image of upright standing DNA origami construct,cf. bottom view of (b). Scale bars=40 nm.

FIG. 2: Characterization of a single hybrid nanopore. (a) Current as afunction of time through a single solid-state nanopore with a diameterof 13 nm. Voltage is switched from 0 mV to +100 mV at 2.8 s. At 6.2 s asudden drop in current indicates insertion of a DNA origami construct,see arrow. (b) Current as a function of time through a single hybridnanopore while reducing the voltage in 10 mV steps. (c) Current-voltagecharacteristic of the hybrid nanopore (line/dots) in comparison with thecurrent-voltage characteristic of the bare solid-state nanopore (plainline).

FIG. 3: Repeated insertion of DNA origami constructs into an 18 nmdiameter solid-state nanopore. (a) Top panel: applied voltage as afunction of time for insertion of DNA origami constructs. At +100 mVhybrid nanopores are stable while at −100 mV the DNA origami constructcan be driven out of the solid-state nanopore. Lower panel:corresponding ionic current trace showing formation of hybrid nanopores,indicated by the arrows. (b) Histogram of the relative current changefor 12 insertions, measured in 5 different experiments and nanopores.The relative current change is the quotient of the hybrid pore currentI_(Hybrid) divided by the open pore current I_(ss). Right: Sketches ofthe situations with I_(ss) and I_(Hybrid).

FIG. 4: λ-DNA events through bare solid-state nanopore and hybridnanopores. (a) Ionic current as a function of time at +100 mV for anexperiment with both DNA origami and A-DNA in the solution, with 0.5 nMand 1.0 nM concentration respectively. For t<11.4 s, DNA translocationsthrough the bare nanopore (15 nm diameter) are observed. After formationof the hybrid nanopore, events for the hybrid nanopore are observed. (b)Typical events for the bare nanopore shown top and the hybrid nanoporeshown bottom. (c) Current histograms indicating DNA translocations forthe bare solid-state and hybrid nanopores. The baseline for bothhistograms was subtracted for easy comparison, and the current wasdigitally filtered at 3 kHz for clarity.

FIG. 5: a schematic representation of a complete testing cycleincluding: (a) the introduction of a DNA construct having a sleeveformation and tail formation into a reservoir on one side of a substratehaving a nanopore; (b) application of a positive polarity potentialdifference to thread the tail formation through the nanopore and seatthe sleeve formation in the nanopore; (c) testing of the currentsignature through the resulting hybrid nanopore when an analyte isintroduced into the reservoir; and (d) expulsion of the construct onapplication of a negative polarity potential difference in readinesse.g. for the introduction of a different DNA construct into thereservoir and a further testing cycle.

FIG. 6: a schematic representation of: (a) the introduction of a DNAconstruct to a substrate having a nanopore, the construct having a bodyformation in the form of a plate defining a central aperture, with atail formation extending from an edge of the aperture; and (b) thedocking of the construct to the substrate, with the tail formationthreaded through the aperture, the flat body formation overlaying thesubstrate, and the central aperture aligned with the nanopore.

FIG. 7: atomic force microscopy pictures of DNA constructs docked to asubstrate (a) and (b) without streptavidin bound to the constructs, and(c) and (d) with streptavidin bound to the constructs.

FIG. 8: schematic plots of the change in ionic current expected ondocking the constructs of FIG. 7 (a) from a solution withoutstreptavidin and (b) from a solution with streptavidin.

FIG. 9: histograms of actual percentage current decrease for repeateddocking experiments using the constructs of FIG. 7 (a) from a solutionwithout streptavidin and (b) from a solution with streptavidin.

In the following we demonstrate the successful insertion of DNAconstructs into solid-state nanopores. Further, we demonstrate that suchconstructs can be controllably and repeatedly inserted into thenanopores and can be used for resistive-pulse sensing. Finally, wedescribe a technique for detecting specific analytes.

Materials and Methods

A DNA origami construct was designed using the square-lattice version ofthe caDNAno software from Douglas et al.¹⁸. As the scaffold strand, the8634 nt-long m13mp18-based^(16a) single strand was chosen and cut at theEcoRI and BamHI restriction sites. Of the 8613 bases of the cutscaffold, 6264 were incorporated into an origami structure with the helpof 142 staple strands and 2349 bases were allowed to form a linearextension of one of the helices. This part of the single-strandedscaffold was complemented with 49 consecutive 40mers and 48mers to forma 2344 basepairs (bp) long double strand with 48 nicks which acted as aleash to ensure correctly-oriented trapping of the DNA construct intothe solid-state nanopore.

The constructs were assembled by heating a Tris-HCl (10 mM)-EDTA (1 mM)buffer (pH 8.0) containing the cut scaffold (10 nM), the staple strandsand the complements of the leash (100 nM each), and 14 mM MgCl₂. Theassembled structures were purified from the excess staple strands byeither agarose gel electrophoresis or centrifugation with 100 kDa MWCOfilters.

Nanopores with diameters between 12-18 nm were fabricated insilicon-nitride (SiN) membranes with an FEI Tecnai F20 transmissionelectron microscope (TEM) equipped with a field emission gun (FEG) whichwas operated at an acceleration voltage of 200 kV and an extractionvoltage of 4000V. The SiN membranes were 30 nm thick with 50×50 μm²windows (DuraSin, Protochips, USA).

The SiN chip containing a nanopore was sealed into polydimethylsiloxane(PDMS) microfluidic channels. The PDMS (Sylgard 184, Dow Corning) wasmade by mixing base and curing agent in a weight ratio of 10:1 andcuring in an aluminum mold for 10 minutes at 150° C. The aluminum molddesign comprised channels at either side of the pore for introducingbuffer and a required sample volume of only 10 μL. The PDMS wasplasma-bonded to a glass slide to prevent leaks¹⁹. Before introducingbuffer, the nanopore containing TEM chip was plasma cleaned for 1minute. A Gigaohm seal was then formed between the two reservoirs of thePDMS by painting fresh PDMS around the edge of the chip and curing on ahot plate for 60 seconds at 120° C. Subsequently, buffer solution wasadded to both sides of the chip.

For ionic current measurements, Ag/AgCl electrodes were fabricated byelectro-deposition of a AgCl layer onto 0.2 μm thick silver wire. Thetwo electrodes were placed in the reservoirs on either side of thenanopore and connected to the headstage of an Axopatch 200B amplifier(Molecular Devices, USA). The amplifier headstage and nanopore devicewere enclosed in a Faraday cage to reduce electromagnetic interference.All ionic current recordings were performed with the internal Besselfilter of the amplifier at 10 kHz and recorded at up to 100 kHzbandwidth. The data was later filtered for analysis. Voltages were setand currents recorded by custom written LabView software^(4,20) or withClampEx (Molecular Devices, USA).

For translocation experiments we used linearized A-DNA (New EnglandBiolabs, USA) diluted in the measurement buffer prior to allexperiments. After purification DNA origami constructs were diluted to aconcentration of approximately 0.5 nM in the measurement buffer.

DNA Origami Construct

An aim of the design process was to find an accessible geometry thatwould fit into the conical form of the solid-state nanopore whileallowing for stable insertion in a wide range of nanopore diameters. Aschematic overview of the construct is shown in FIG. 1. All lengths arecalculated by assuming 0.34 nm helical step size per bp and an averagecenter-to-center distance of 2.5 nm per helix pair (see FIG. 1b)^(15,21).

The geometry has a sleeve formation of staggered double helices whosez-axes are pointing into the solid-state pore forming four overlappingskirts with a quadratic base, the helices defining a central aperture.The innermost skirt is formed by 16 helices, 48 bp long (16.3 nm), whereeach face of this square cylinder is comprised of five parallel helicesresulting in an outer edge length of 12.5 nm and an inner edge length of7.5 nm, see FIG. 1b . The next skirt is built up from 24 helices (7helices per face) and overlaps with the innermost skirt over a length ofat least 16 bp to a maximum of 40 bp. The third layer consists of 32helices and the outermost layer of 40 helices with each layeroverlapping over at least 8 bp to a maximum of 16 bp. For the edgefacing the solution (distal) we calculated a length of 11×2.5 nm=27.5 nmwhich is large enough to prevent slipping of the assembled structureinto the solid-state pore, which in our experiments has a diameter oftypically about 12-18 nm. In order to guide the voltage-drivenself-assembly, we added a double stranded DNA tail to the tip of thesleeve formation. It is believed that the tail threads through thenanopore to assist and precede the insertion of the sleeve formationtherein. The overall length of the origami structure without the tail is51.0 nm, the outer diagonal diameter is 38.9 nm, while the centreopening is 7.5×7.5 nm². Typical TEM images recorded at 100 kV (JEM-1011,JEOL) of the correctly folded structure are shown in FIGS. 1c and 1d ,as side and top view, respectively. The segmented double stranded DNAtail can be observed in both images.

Hybrid Nanopore Formation

After a solid-state nanopore was assembled into a microfluidicmeasurement cell, the current-voltage characteristic was first tested ina 20 nm filtered buffer solution of 0.5×TBE, 5.5 mM MgCl₂, 1M KCl. FIG.2 shows an example trace of the characteristic current signatureobserved after adding the DNA origami construct to one side of thesolid-state nanopore. Recording in this experiment started at 0 mV andafter 2.8 s+100 mV was applied (with positive polarity making the DNAconstruct move towards the solid-state nanopore). An initial ioniccurrent of 8.7 nA was measured through the bare solid-state nanopore.After about 6.6 s we see a sudden drop in the ionic current indicatingthe correct insertion of a DNA origami construct into the solid-statenanopore. The newly formed hybrid nanopore was then tested by recordinga current-voltage characteristic shown in FIG. 2b . The voltage wasdecreased from +100 to −100 mV in steps of −10 mV. The points shown inFIG. 2c are extracted by averaging over each current step. The line forthe current-voltage characteristic of the bare solid-state nanopore wasmeasured before adding the DNA origami solution. We observe that atpositive voltages the current through the hybrid nanopore is lower thanfor the bare solid-state nanopore. The bare solid-state nanopore shows alinear IV curve whereas after the formation of a hybrid nanopore the IVcurve shows rectification. This behavior is due to the asymmetry and thehighly charged DNA surface of the hybrid nanopore²², and is an importantindication that the DNA origami structure is correctly assembled intothe solid-state nanopore.

Repeated Construct Insertions

Our DNA origami constructs can be ejected from the solid-state nanoporeby sudden reversal of the applied potential. This is an importantfeature allowing for error correction if a pore did not insert correctlyor for exchanging different pores within the same experiment. The toppart of FIG. 3a shows the applied voltage as a function of timeswitching between +100 and −100 mV. The corresponding ionic currenttrace is shown in the lower part of FIG. 3a . Initially, at +100 mV, ahybrid nanopore is present and a current of 10.2 nA is observed. Uponswitching the voltage to −100 mV the current changes to −11.8 nA, butswitching back to +100 mV leads to an increase of the current to +11.6nA indicating the original open pore current and the disassembly of thehybrid nanopore. At this voltage the same or another construct isquickly inserted into the solid-state nanopore at 11.0 s (see arrow inFIG. 3a ). The current stabilizes at approximately the same level as forthe previous hybrid nanopore. At t=21.6 s the voltage is switched againto −100 mV to eject the origami structure. Upon switching back to +100mV at 26.6 s the ionic current returns quickly to the open nanoporecurrent only to fall back to the hybrid nanopore current of 10.0 nA at31.0 s, indicating another successful assembly.

The histogram in FIG. 3b shows the relative current change defined asthe hybrid nanopore current divided by the solid-state nanopore currentat +100 mV for 12 insertions taken for 5 different nanopores. Insertionof the origami construct reduced the solid-state nanopore current toaround 80% of its initial value. This is expected as counter ions in thevicinity of the DNA are mobile²³. We see a consistent change in ourexperiments with solid-state nanopores with a range of diameters whichpoints to a well-controlled insertion of the DNA origami structure.

Our results show that we can controllably and repeatedly insert andeject DNA origami constructs.

Detection of DNA with Hybrid Nanopores

We are able to detect λ-DNA strands in our hybrid nanopores. In thisexperiment DNA origami constructs with a concentration of 0.5 nM wereadded in addition to 1 nM of λ-DNA solution. As shown in FIG. 4a , weobserve the typical signatures for long DNA molecules translocating abare solid-state nanopore, indicating DNA forming a tight hairpin andfolding onto itself^(4a,24). After insertion of a DNA origami constructat 11.4 s (FIG. 4a ), the current stabilizes after a few seconds albeitwith a higher level of noise. Since the λ-DNA is present in thebackground, λ-DNA interactions with the hybrid nanopore will occur.Three typical events are presented for DNA translocations in FIG. 4b forthe bare and hybrid nanopores, respectively. The corresponding currenthistograms are shown in FIG. 4c . We observe typical DNA translocationevents as peaks in the bare solid-state pore current histogram (FIG. 4c) with peaks at −100 pA and −200 pA. The data is shown after thebaseline was subtracted. The peaks indicate that DNA folding is readilyobservable in our bare solid-state nanopore prior to hybrid nanoporeformation. For the hybrid nanopore current histogram, we observe a clearpeak at roughly −60 pA, while we also detect a number of folded eventsalthough with lower frequency (FIGS. 4b and 4c ). This can be explainedby the considerably smaller diameter of the hybrid nanopore.

FIG. 5 shows schematically a complete testing cycle including: (a) theintroduction of a DNA construct 1 having a sleeve formation 2 and tailformation 3 into a reservoir 4 on one side of a substrate 5 having ananopore 6; (b) application of a positive polarity potential differenceto thread the tail formation through the nanopore and thereby dockingthe construct to the substrate with the sleeve formation seated in thenanopore and the central aperture of the sleeve aligned with thenanopore; (c) testing of the current signature through the resultinghybrid nanopore when an analyte 7 is introduced into the reservoir; and(d) ejection of the construct on application of a negative polaritypotential difference in readiness e.g. for the introduction of adifferent DNA construct into the reservoir and a further testing cycle.

CONCLUSIONS

We have experimentally demonstrated the formation of hybrid nanoporescomprising 3D DNA origami structures inserted into a solid-statenanopore. The hybrid nanopores can be repeatedly assembled by reversingthe applied potential, which enables the functionality of a nanoporesensor to be changed during an experiment. Our measurements of DNAdetection show that DNA origami constructs can be used asresistive-pulse sensors. The constructs offer the possibility to adaptthe diameter, shape and surface functionality of hybrid

However, although the concept of construct insertion and expulsion hasbeen demonstrated using a DNA construct, it could also be performedusing constructs based on other suitable molecules. In particular, amolecular construct which, like DNA, can provide a hydrophilic externalsurface, may similarly help to prevent the construct from binding tootightly to the mating surface of the nanopore.

Further, although we have used a construct having a sleeve formation(which shows particular promise for stable docking and for controllingthe functionality of the nanopore), other types of construct can beadopted. For example, FIG. 6 shows schematically (a) the introduction ofa DNA construct 11 to a substrate 15 having a nanopore 16, the DNAconstruct having a body formation in the form of a flat plate 12defining a central aperture 18, with a tail formation 13 extending froman edge of the aperture, and (b) the docking of the construct to thesubstrate, with the tail formation threaded through the aperture, theflat body formation overlaying the substrate, and the central aperturealigned with the nanopore.

Specific Analyte Detection

The approach described above involves measuring changes in ionic currentas molecules translocate through the nanopore. Molecules of differenttype but similar molecular mass tend to generate similar changes inionic current as the molecules translocate through the nanopore. Thus wenext describe an approach which provides detection specificity forparticular analytes.

More particularly, a molecular construct such as a DNA origami constructdescribed above, can be used to trap an analyte of interest at the mouthof the nanopore by introducing one or more binding sites for theparticular analyte at the aperture in the construct. Each time aconstruct is docked to the nanopore a current decrease due to theconstruct partially blocking the ionic current flow (FIG. 1) through thepore can be measured. However, when the analyte is present in thesolution and therefore bound at the aperture, the current decrease istypically greater than when the analyte is absent from the solution.

This detection method can be adapted to many biomolecules of interest,such as antibodies, since it only requires the provision of bindingsites on the construct, which in the case of DNA constructs can beachieved readily with oligonucleotide modifications offered by DNAsynthesis companies.

The system is reversible. For example, in the case of DNA constructs anda SiN chip containing nanopores, a high positive voltage can be appliedto dock the constructs to the pores and a high negative voltage can beapplied to force them away from the pores. The step change in ioniccurrent upon construct docking can be measured many times in oneexperiment (e.g. over hundreds of voltage reversals) to build up robuststatistics. Further, since only a step change in current is beingmeasured, low bandwidth electronics can be used.

As a proof of principle, a flat square DNA origami construct with acentral aperture and four binding sites at the mouth of the aperture forthe protein streptavidin was designed. A solution was preparedcontaining 0.5×TBE, 5.5 mM MgCl₂, 1M KCl. The constructs were then addedto the solution, which was introduced to a SiN chip similar to thosedescribed above. A positive potential was applied to dock the constructsto the nanopores of the chip. Although the DNA constructs did not havetail formations, the apertures of the constructs were nonetheless ableto align to the pores of the chips. FIGS. 7(a) and (b) show atomic forcemicroscopy pictures of the docked constructs.

The procedure was then repeated but with streptavidin added to thesolution. FIGS. 7(c) and (d) show atomic force microscopy pictures ofthe docked constructs, with bound streptavidin clearly visible at theapertures.

FIG. 8 shows schematically the change in ionic current that would beexpected on docking of the constructs (a) from the solution withoutstreptavidin and (b) from the solution with streptavidin. The expecteddecrease in current is greater for (b) than (a). The solution withoutstreptavidin thus provides a reference decrease against which thedecrease from a further solution which may contain streptavidin can becompared. If the decrease associated with the further solution differsfrom the reference by an amount similar to the amount by which (b)differs from (a), then that is an indication that the further solutioncontains streptavidin.

FIGS. 9(a) and (b) are histograms of percentage current decrease foractual experiments using the flat square DNA origami construct andstreptavidin system of FIGS. 7(a) to (d). For the histogram of FIG. 9(a)the current decrease for the construct docking process using thereference system without any protein attached (FIGS. 7(a) and 7(b)) wasrepeatedly measured, whereas for the histogram of FIG. 9(b) the currentdecrease for the construct docking process using the system with theprotein attached (FIGS. 7(c) and 7(d)) was repeatedly measured. Asexpected, the reference system shows a lower reduction in current thanthe system with the streptavidin bound to the construct. To ensure thatthe histograms were directly comparable, both the reference and thestreptavidin-bound constructs were docked to the samenanopore-containing chip.

While the invention has been described in conjunction with the exemplaryembodiments described above, many equivalent modifications andvariations will be apparent to those skilled in the art when given thisdisclosure. Accordingly, the exemplary embodiments of the invention setforth above are considered to be illustrative and not limiting. Variouschanges to the described embodiments may be made without departing fromthe spirit and scope of the invention.

All references are hereby incorporated by reference.

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The invention claimed is:
 1. A system comprising: a substrate containingone or more nanopores through the substrate; and one or more respectivepolynucleotide constructs which are docked to the one or more nanopores,each construct having a DNA body formation in the form of a flat platedefining a central aperture; and a tail formation extending from thebody formation and threading through its nanopore to dock the constructto the substrate with the aperture and nanopore in alignment.
 2. Asystem according to claim 1, wherein the tail formation extends from anedge of the aperture.
 3. A system according to claim 1, wherein the bodyformation overlays the substrate.
 4. A system according to claim 1,wherein the tail formation comprises double-stranded DNA.
 5. A systemaccording to claim 1, wherein the body formation has a docking surfacewhich is hydrophilic.
 6. A system according to claim 1, wherein theconstruct includes at least one of functional chemical groups,fluorophores, and gold nanoparticles.
 7. A system according to claim 1,wherein the substrate is silicon-nitride or silicon-oxide.
 8. A systemaccording to claim 1, wherein the diameter of the one or more nanoporesis 100 nm or less.
 9. A system according to claim 1, wherein thediameter of the one or more nanopores is 1 nm or more.
 10. A systemaccording to claim 1, wherein the flat plate is square.
 11. A method fordetermining the presence of an analyte, comprising the steps of:providing a system according to claim 1; measuring ionic current flowthrough the nanopores while translocating, analyte moleculestherethrough; and determining that the analyte molecules are presentwhen a measured change in ionic current differs from a reference changein ionic current by a predetermined amount.
 12. A method of controllingthe functionality of a substrate containing at least one nanopore, themethod including the steps of: introducing to the substrate a solutioncontaining a polynucleotide construct having a DNA body formation in theform of a flat plate defining a central aperture, and a tail formationextending from the body formation; applying a potential differenceacross the substrate to thread the tail formation through the nanoporethereby docking the molecular construct to the substrate with theaperture and nanopore in alignment; and expelling the polynucleotideconstruct from the substrate by varying the potential difference.
 13. Amethod according to claim 12, wherein the DNA body formation has adocking surface which contacts the substrate when the construct isdocked thereto, at least the docking surface being hydrophilic.
 14. Amethod according to claim 12, including further steps after theexpelling step of: introducing to the substrate a solution containing adifferent polynucleotide construct having a DNA body formation in theform of a flat plate defining a central aperture, and a tail formationextending from the body formation; and re-applying a potentialdifference across the substrate to thread the tail formation through thenanopore thereby docking the different polynucleotide construct to thesubstrate with the aperture and nanopore in alignment.
 15. A methodaccording to claim 12, wherein the tail formation extends from an edgeof the aperture.
 16. A method according to claim 12, wherein the bodyformation overlays the substrate.
 17. A method according to claim 12,wherein the tail for on comprises double-stranded DNA.
 18. A method ofdetecting the presence of an analyte in a solution, the method includingthe steps of: (a) providing a substrate containing at least onenanopore; (b) introducing to the substrate a solution which may containthe analyte, and a polynucleotide construct having a DNA body formationin the form of a flat plate defining a central aperture, and a tailformation extending from the body formation, and which has a one or morebinding sites for the analyte at the aperture; (c) applying a potentialdifference across the substrate to dock the polynucleotide construct tothe substrate with the aperture and nanopore in alignment, and measuringthe corresponding change in ionic current through the nanopore; (d)comparing the measured change in ionic current to a reference change inionic current; and (e) determining that the analyte is present in thesolution when the measured change in ionic current differs from thereference change in ionic current by more than a predetermined amount.19. A method according to claim 18, wherein the applying step (c)includes repeatedly performing the sub-steps of: (i) applying thepotential difference across the substrate to dock the polynucleotideconstruct to the substrate with the aperture and nanopore in alignment,and measuring the corresponding change in ionic current through thenanopore; and (ii) expelling the polynucleotide construct from thesubstrate by varying the potential difference; and wherein, in thecomparing step (d), the measured change in ionic current is an averagemeasured change derived from the repeated measurements of sub-step(c-i).
 20. A method according to claim 18, further including the stepsof: (A) providing a substrate containing at least one nanopore; (B)introducing to the substrate a solution containing the polynucleotideconstruct in the absence of the analyte; and (C) applying a potentialdifference across the substrate to dock the polynucleotide construct, inthe absence of the analyte, to the substrate with the aperture andnanopore in alignment, and measuring the corresponding change in ioniccurrent through the nanopore; and wherein, in the comparing step (d),the reference change in ionic current is the change in current measuredat step (C).
 21. A method according to claim 20, wherein the applyingstep (C) includes repeatedly performing the sub-steps of: (i) applyingthe potential difference across the substrate to dock the polynucleotideconstruct, in the absence of the analyte, to the substrate with theaperture and nanopore in alignment, and measuring the correspondingchange in ionic current through the nanopore; and (ii) expelling thepolynucleotide construct from the substrate by varying the potentialdifference; and wherein, in the comparing step (d), the reference changein ionic current is an average measured change derived from the repeatedmeasurements of sub-step (C-i).
 22. A method according to claim 18,wherein the analyte is DNA or an antibody.
 23. A method according toclaim 18, wherein the tail formation extends from an edge of theaperture.
 24. A method according to claim 18, wherein the body formationoverlays the substrate.
 25. A method according to claim 18, wherein thetail formation comprises double-stranded DNA.
 26. A method of docking apolynucleotide construct to a substrate having a nanopore, the constructhaving a DNA body formation in the form of a flat plate defining acentral aperture, and a tail formation extending from the bodyformation, the method including the steps of: introducing to thesubstrate a solution containing the polynucleotide construct; andapplying a potential difference across the substrate to thread the tailformation through the nanopore thereby docking the molecular constructto the substrate with the aperture and nanopore in alignment.
 27. Amethod according to claim 26, wherein the tail formation extends from anedge of the aperture.
 28. A method according to claim 26, wherein thebody formation overlays the substrate.
 29. A method according to claim26, wherein the tail formation comprises double-stranded DNA.